In earlier work, we found that effective in vitro activation of naive CD8 T cells requires three signals. TCR and CD28 signals are sufficient to stimulate proliferation, but a signal provided by IL-12 or Type I IFN (IFN1/2) is required to stimulate differentiation. During the current granting period peptide immunization, tumor and pathogen models have provided a great deal of evidence in support of this three signal model. We have also found that IL-12 and IFN1 induce a gene regulation program that results in up-or down- regulation of about 350 genes, superimposed on the regulation that occurs in response to TCR and CD28 signals. A number of transcription factors are regulated by IL-12/IFN1, including T-bet and eomesodermin (Eomes). In addition, IL- 12/FN1 upregulate expression of B lymphocyte-induced maturation protein-1 (Blimp-1), the 'master regulator'of terminal B cell differentiation, and downregulate a number of other transcription repressors, including Bcl-6. Preliminary results strongly support the hypotheses that: 1) CD8 T cells initiate the differentiation program in response to 2 signals, but in the absence of a third signal programming is terminated as a result of upregulated expression of multiple transcriptional repressors, and 2) Blimp-1, and its counter-repressor Bcl-6, play central roles in signal 3-dependent regulation of gene expression in Ag-activated CD8 T cells, with Blimp-1 acting to repress expression of transcriptional repressors that terminate differentiation. Thus, Blimp-1 may act as a 'master regulator'in CD8 T cell differentiation and Bcl-6 may have a role in regulating development of memory, somewhat analogous to their roles in formation of plasma cells and memory B cells. Two Specific Aims are proposed to test these and additional hypotheses: Specific Aim 1. To determine the role of Blimp-1 in regulating gene expression for signal 3-dependent CD8 T cell programming. The roles of Blimp-1 in regulating IL-12/IFN1-dependent changes in gene expression will be examined in both in vitro and in vivo models. We will focus on roles for Blimp-1 in regulating Bcl-6 and other transcription repressors, and the positive transcription factors T-bet and Eomes. Specific Aim 2. To determine the roles of Blimp-1/Bcl-6 in regulating gene expression for CD8 T cell differentiation and memory formation. Bcl-6 is likely to have a key role in regulating CD8 T cell differentiation, at least in part through its role as a counter-repressor of Blimp-1, and this will be examined. Furthermore, the balance of Blimp-1/Bcl-6 expression may regulate the transition to memory, and may determine whether an activated CD8 T cell becomes an effector memory cell or a central memory cell. Several hypotheses regarding Blimp-1/Bcl-6 roles in this transition will be tested. We anticipate that the planned experiments will provide significant new insights into the molecular regulation of the CD8 T cell differentiation program, and that the results will have implications for the design of prophylactic and therapeutic vaccines that target CD8 T cell responses. PUBLIC HEALTH RELEVANCE: CD8 T lymphocytes, or cytotoxic T lymphocytes, are an important arm of immune system defense against bacterial and viral infections, and can potentially be effective in controlling cancers. The studies supported by this grant will determine how the signals that activate these cells regulate patterns of gene expression that program the cells to carry out their functions and become protective memory cells following an infection or vaccination. The findings will contribute to a more rational basis for manipulating responses with vaccines for protection and therapy of infections and cancers, and are likely to provide novel strategies for accomplishing this.